Tryptophan synthase of Escherichia coli and of Salmonella typhimurium is composed of two Alpha subunits and a Beta dimer. This bienzyme complex is a good model for more complex multienzyme complexes. The separate Alpha and Beta subunits each catalyze their own characteristic reactions; the rate of each reaction is greatly altered by the presence of the complementary subunit. We are investigating the structure and function of the separate Alpha and Beta subunits in order to understand the mechanism of the reactions catalyzed by each subunit and to determine how subunit interaction affects the catalytic and structural properties of each subunit. We have identified and located in the sequence of the Beta subunit several active site residues, including, most recently, an arginyl residue (Arg-148) which binds the carboxyl residue of the substrate L-serine. A reactive arginyl residue in the Alpha subunit may bind the phosphate group of the substrate indole glycerol phosphate. The active site mechanisms of the Alpha and Beta subunits are being investigated by additional chemical modification studies and by spectroscopic studies using substrate analogs and effinity labels. We are also investigating the folding the domain structure of the Alpha subunit of tryptophan synthase from Escherichia coli, from Salmonella typhimurium, and from an interspecies hybrid. This hybrid has one domain which originates from S. typhimurium and one domain which originates from E. coli. We have compared the guanidine hydrochloride-induced denaturation and thermal denaturation of these three Alpha subunits by use of circular dichroism measurements. The two domains of each Alpha subunit are unfolded by guanidine hydrochloride in a stepwise fashion. Our finding that the stability of each of the two domains of the interspecies hybrid is similar to the stability of the corresponding domain in the species from which it originates gives information on the evolution and divergence of protein structures.